The RNA interference(RNAi)-mediated gene silencing has become an important technology for manipulating cellular phenotypes, mapping genetic pathways and discovering therapeutic targets, and has therapeutic potential. A key challenge to realizing the broad potential of RNAi technology is the need for safe and effective methods for delivery Small interfering RNA (siRNA) into targeted cell. Although a variety of materials and approaches have been explored for siRNA delivery, the performance of current materials and approaches remain unsatisfactory because they generally do not work well with primary cells or non-adherent cell types and often result in some degree of cytotoxicity and alterations in cell biology, thereby severely limiting the cell types amenable to discovery research and not suitable for human therapeutics. In this project, we propose to investigate a novel, molecularly designed multi-functional DNA Origami nanostructures (DONs) for effective delivery of siRNA inside targeted cells. Our hypothesis is that the biocompatible, molecularly defined, monodispersed DONs incorporated with siRNAs, and cell-penetrating peptides (CPP) would enables effective siRNA uptake by cells and efficient release of siRNA inside cells, resulting in high gene knockdown even when using low siRNA concentrations. Our ultimate goal is to develop a safe and effective DONs-siRNA therapeutic for diseases; the short-term goal of this Phase I project is to develop DONs-based multi-functional siRNA delivery and transfection reagent with optimal structure to aid siRNA silencing research. The Phase I proof-of-concept demonstration project will focus on 1) developing chemistry and protocol for constructing DONs-siRNA and 2) in vitro assessing the effectiveness of DONs-siRNA for cell transfection and gene knockdown. We plan to develop one-pot approach to create multi-functional DONs incorporated with firefly luciferase(Luc) siRNA gene and green fluorescent protein (GFP) siRNA gene, and evaluate their silencing efficiency to Luc-expressed HeLa cells and GFP-expressed Human prostate carcinoma (PC-3) cells. In Phase II we will thoroughly optimize the preparation of multi-functional DON-siRNA incorporated with cell-target ligands, investigate the in vivo delivery of DON-siRNA and the effectiveness of gene knockdown, the pharmacokinetic profile and organ biodistribution of DON-siRNA in nude mice bearing tumors.